Native human growth hormone is a single polypeptide chain protein consisting of 191 amino acids, internally cross-linked by two disulphide bridges. The growth hormones of other animal species are closely homologous to native human growth hormone, and have similar biological activity in terms of being effective in the treatment of diseases related to growth hormone deficiencies in humans, such as hypopituitary dwarfism and osteoporosis. Recombinant forms of human growth hormone have also been produced with the same or substantially similar amino acid sequence as native human growth hormone and identical biological activity to the native hormone. Except as otherwise noted below, all native and recombinant forms of human growth hormone are collectively referred to as “hGH.” Because of the structural similarities between hGH and the growth hormones of other species, one would expect formulations that are effective in stabilizing hGH to also be effective in stabilizing the growth hormones of other species.
hGH is primarily sold in lyophilized form today. See, for example, GENOTROPIN® Lyophilized Powder (Pharmacia & Upjohn Company, now owned by Pfizer Inc.), HUMATROPE® (Eli Lilly), NORDITROPIN® for Injection (Novo Nordisk), SAIZEN® for Injection (Serono), and NUTROPIN® (Genentech). Lyophilized formulations have the advantage of providing protein stability for long periods of time. However, a lyophilized formulation must be used shortly after reconstitution, as aggregation and deamidation tend to begin shortly after reconstitution with an aqueous diluent. This generally means that it is left up to the consumer of a lyophilized formulation to reconstitute the product shortly before use. If reconstitution is not done properly, or if the reconstituted formulation is stored for too long before use, the consumer may get an improper dosage of the growth hormone or a dosage containing unsuitable levels of hGH degradation products. In addition, manufacture of lyophilized formulations involves substantially greater cost and time relative to manufacture of liquid formulations.
Due to the above disadvantages with lyophilized hGH formulations, various liquid formulations of hGH have been developed over the years, each with varying degrees of stability under various storage and handling conditions. Two commercial liquid formulations of hGH are sold under the brand names NUTROPIN AQ® (Genentech, Inc.) and NORDITROPIN® (Novo Nordisk). The composition of these and other hGH liquid formulations have been disclosed in issued patents and published patent applications, summarized below. Each of the following references states that the liquid formulations of hGH formulations described therein are stable at refrigeration temperatures, at about 2° C. to about 8° C., except where noted otherwise below. However, none claim to disclose formulations that can withstand exposure to freezing and thawing, conditions to which products can be exposed in transit. Furthermore, the hGH in some of the previously disclosed formulations degrades or undergoes aggregation when subjected to physical agitation, for example, during shipment. These types of instabilities not only cause wastage of expensive product, but can also cause safety issues if the degraded product is inadvertently administered to a patient.
U.S. Pat. No. 5,567,677 (invented by Castensson et al.; assigned to PHARMACIA AB) discloses an aqueous formulation consisting of growth hormone and citrate buffer in an amount of 2-50 mM at a pH of about 5.0 to 7.0. The '677 patent also teaches that mannitol and glycine can be suitably included in the formulation disclosed therein.
U.S. Pat. Nos. 5,763,394 and 5,981,485 (invented by O'Connor et al.; assigned to GENENTECH, INC.) disclose an aqueous human growth hormone formulation containing hGH, a buffer providing pH 5.5 to pH 7 (e.g., sodium citrate), 0.1% to 1% w/v non-ionic surfactant (e.g., polysorbate 20) and, 50 to 200 mM of a neutral salt (e.g. sodium chloride), and a preservative (e.g. phenol), wherein said formulation is free of glycine and mannitol.
U.S. Pat. No. 6,022,858 (invented by Sorensen et al., assigned to NOVO NORDISK A/S), discloses a buffered aqueous solution containing a human growth hormone pretreated with zinc salt, and optionally containing lysine or calcium ion.
U.S. Pat. No. 5,849,704 (invented by Sorensen et al., assigned to NOVO NORDISK A/S), discloses a buffered aqueous solution containing a growth hormone buffered with histidine or histidine derivative.
U.S. Pat. No. 5,977,069, U.S. Pat. No. 5,631,225, and U.S. Pat. No. 5,547,696 (invented by Sorensen et al., assigned to NOVO NORDISK A/S), disclose buffered aqueous solutions containing a human growth hormone with stabilizing amounts of the amino acids asparagine, isoleucine, or valine, respectively.
U.S. Pat. No. 5,705,482 and U.S. Pat. No. 5,552,385 (invented by Christensen et al., assigned to NOVO NORDISK A/S), disclose buffered aqueous solutions containing a human growth hormone with stabilizing amounts of the peptides Leu-His-Leu and Lys-Gly-Asp-Ser respectively.
WO 01/03741 A1 (for an invention by Siebold et al.; assigned to GRANDIS BIOTECH GMBH) discloses a “storage stable liquid growth hormone formulation consisting essentially of growth hormone in isotonic phosphate buffered solution” and also claims formulations with phosphate buffer and a non-ionic surfactant present at a concentration of 0.2% or less. In the Examples section of the publication, the only non-ionic surfactant used is Pluronic F-68 at a concentration of 0.2% (w/v) in each of the formulations where it was included.
WO 02/067989 A1 (for an invention by Seibold et al.; assigned to GRANDIS BIOTECH GMBH) is directed to “an aqueous growth hormone formulation comprising growth hormone and (a) citrate buffer of about pH 5.6 or more, or (b) a buffer other than citrate of about pH 6.0 or more, and substantially free of crystallization on storage.” The only suitable temperatures for storage of the disclosed formulations are refrigeration temperature (4° C. to 8° C.) and above, or in a temperature range of 8° to 25° C.
U.S. Application Publication No. 2002/0077461 (for an invention by Bjorn et al.; assigned to NOVO NORDISK OF NORTH AMERICA INC.) discloses pharmaceutical formulations comprising growth hormone (e.g., hGH), an amino acid selected from the group consisting of asparagine, isoleucine, valine, leucine, histidine, a derivative of histidine, or a peptide comprising at least one basic amino acid residue and at least one acidic amino acid residue, and a non-ionic detergent (e.g., a polysorbate or a polyaxamer). The application also discloses such formulations with a buffer, (e.g. histidine, citrate, tartrate, or phosphate) for pH 6 to pH 8, a tonicity agent (e.g., mannitol). The only stability studies disclosed in this application were carried out at refrigerated temperatures or above.
WO 01/24814 A1 (for an invention by Chen et al.; assigned to CHIRON CORPORATION) discloses the use of an amino acid base sufficient to decrease aggregate formation during storage to stabilize aqueous polypeptide formulations, where the amino acid base comprises at least one amino acid selected from the group consisting of arginine, lysine, aspartic acid, and glutamic acid. This published application also discloses the inclusion of additional stabilizers in such formulations, including antioxidants, such as methionine, and non-ionic surfactants. Interleukin-2 is the only polypeptide whose stabilization using such a formulation is illustrated therein.
The commercial formulations of liquid hGH presently available on the market include phenol as a preservative. See, for example, NUTROPIN AQ® (a liquid formulation of recombinant hGH sold by Genentech Inc.), and NORDITROPIN® (a liquid formulation of recombinant hGH sold by Novo Nordisk). However, phenol is known to promote the aggregation of hGH, especially upon freezing and thawing (see Maa, Yuh-Fun, et al., Internat J Pharm 140: 155-168 (1996)).
Even with all the advancements that have been made to date in the development of formulations that stabilize growth hormones in particular and polypeptides in general, the stability of hGH in liquid formulations remains a problem. Stability is particularly problematic in liquid hGH formulations exposed to freezing and subsequent thawing, especially when a phenolic excipient (e.g. phenol preservative) is present. Even a single freeze-thaw can render known liquid hGH formulations, such as the commercial formulations cited above, unsuitable for human use, due to protein aggregation and precipitate formation.
There is a need for a liquid hGH formulation that remains stable under freeze-thaw conditions, as well as under other conditions of physical stress, such as physical agitation, provided the formulation also remains stable after long term storage, under suitable storage conditions. Such a formulation could be stored not only in a refrigerator, as are the current commercial hGH formulations, it could also be stored in a freezer.